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Abstract

Background

The prevalence of type 2 diabetes (T2D) is increasing worldwide and creating a significant
burden on health systems, highlighting the need for the development of innovative
therapeutic approaches to overcome immune dysfunction, which is likely a key factor
in the development of insulin resistance in T2D. It suggests that immune modulation
may be a useful tool in treating the disease.

Methods

In an open-label, phase 1/phase 2 study, patients (N = 36) with long-standing T2D
were divided into three groups (Group A, oral medications, n = 18; Group B, oral medications
+ insulin injections, n = 11; Group C having impaired β-cell function with oral medications
+ insulin injections, n = 7). All patients received one treatment with the Stem Cell
Educator therapy in which a patient’s blood is circulated through a closed-loop system
that separates mononuclear cells from the whole blood, briefly co-cultures them with
adherent cord blood-derived multipotent stem cells (CB-SCs), and returns the educated
autologous cells to the patient’s circulation.

Conclusions

Clinical data from the current phase 1/phase 2 study demonstrate that Stem Cell Educator
therapy is a safe approach that produces lasting improvement in metabolic control
for individuals with moderate or severe T2D who receive a single treatment. In addition,
this approach does not appear to have the safety and ethical concerns associated with
conventional stem cell-based approaches.

Trial registration

Background

Type 2 diabetes (T2D) is a major global health issue, with prevalence rates exceeding
12.1% of the population in India, 9.7% in China, and 8.3% in the United States [1,2]. According to a report from the American Diabetes Association (ADA, Philadelphia,
PA, USA), the total number of Americans living with diabetes will increase 64% by
2025, and diabetes-related Medicare expenditures will increase by 72% to $514 billion/year.
Moreover, diabetes and its associated complications (for example, cardiovascular diseases,
stroke, kidney failure and poor circulation) markedly decrease the quality of life,
limiting the regular activity and productivity of individuals with the disease and
creating significant economic and social burdens [3]. Thus, it is a top priority to find a cure for T2D. To date, animal and clinical
studies demonstrate that insulin resistance is the key mechanism leading to the development
and pathogenesis of T2D, though many factors are known to contribute to the development
and severity of the disease (for example, obesity, genetic factors and sedentary lifestyle)
[3]. Several medications have been shown to improve the outcome of T2D treatment through
various mechanisms and act on various organs and tissues. However, safety concerns
limit the utility of known insulin sensitizers. For example, the peroxisome proliferator-activated
receptor-γ (PPAR-γ) agonists (thiazolidinediones, TZDs) are some of the major frontline
insulin-sensitizing drugs for clinical treatment of T2D that directly improve insulin
sensitivity, but the risk of adverse effects with long-term use of these compounds
is a safety concern [4,5]. Alternative approaches are needed.

Increasing evidence reveals that T2D subjects display multiple immune dysfunctions
and chronic metabolic inflammation. Specifically, inflammatory cytokines derived from
adipocytes and macrophages promote the development of insulin resistance in T2D through
JNK and/or IKKβ/NF-κB pathways, including changes in the levels of tumor necrosis
factor-α (TNFα), interleukin-1 (IL-1), IL-6, IL-17, monocyte chemoattractant protein-1
(MCP-1), resistin and plasminogen activator inhibitor-1 (PAI-1) [6-10]. Control or reversal of these immune dysfunctions and chronic inflammation may provide
an alternative approach for overcoming insulin resistance and may point to a cure
for diabetes. However, the failure of several recent clinical trials in Type 1 diabetes
(T1D) highlights the challenges we face in conquering the multiple immune dysfunctions
by using conventional immune approaches in humans [11-13]. Based on pre-clinical studies in mice and humans [14-17], we have developed Stem Cell Educator therapy [18], an innovative technology designed to control or reverse immune dysfunctions. Stem
Cell Educator therapy consists of a closed-loop system that circulates a patient’s
blood through a blood cell separator (MCS+, Haemonetics, Braintree, MA, USA), briefly
co-cultures the patient’s lymphocytes with adherent cord blood-derived multipotent
stem cells (CB-SCs) in vitro, and returns the educated lymphocytes (but not the CB-SCs) to the patient’s circulation
[18]. Our initial clinical trial in T1D revealed that a single treatment with the Stem
Cell Educator provides lasting reversal of immune dysfunctions and allows regeneration
of islet β cells and improvement of metabolic control in subjects with long-standing
T1D [18,19]. Here, we explore the therapeutic potential of Stem Cell Educator therapy in T2D
subjects.

Methods

Patients

T2D subjects receiving care through the Section of Endocrinology at the General Hospital
of Jinan Military Command (Jinan, Shandong, China) were enrolled in a phase 1/phase
2, open-label clinical trial conducted from August 2011 through September 2012. With
oversight from a planning committee, the principal investigator designed the trial
and received ethical approval for the clinical treatment protocol and consent from
the General Hospital of Jinan Military Command. Written informed consent was obtained
from each participant. All subjects receiving Stem Cell Educator therapy had been
treated with diet, exercise, oral medications and/or insulin injections at stable
doses for at least six months prior to treatment. Key exclusion criteria included
clinically significant liver, kidney or heart disease; pregnancy; immunosuppressive
medication; viral diseases; or diseases associated with immunodeficiency; or any other
clinically significant, coexisting conditions.

Stem Cell Educator therapy and follow-up

In an open-label, phase 1/phase 2 study, patients (N = 36) with long-standing T2D
were divided into three groups (Group A, oral medications, n = 18; Group B, oral medications
+ insulin injections, n = 11; and Group C having impaired islet β cell function with
oral medications + insulin injections, n = 7). Thirty-six participants received a
single treatment with the Stem Cell Educator (Tianhe Stem Cell Biotechnology®). The
preparation of CB-SC cultures and Stem Cell Educators were performed as previously
described [18]. Briefly, a 16-gauge IV needle was placed in the left (or right) median cubital vein,
and the patient’s blood was passed through a blood cell separator MCS+ (Haemonetics®,
Braintree, MA, USA) for six to seven hours to isolate mononuclear cells in accordance
with the manufacturer’s recommended protocol. The collected mononuclear cells were
transferred into the device for exposure to allogeneic CB-SCs. CB-SC-treated mononuclear
cells were returned to the patient’s circulation via a dorsal vein in the hand with
physiological saline. The whole process takes eight to nine hours. Follow-up visits
were scheduled 4, 12, 24, 40 and 56 weeks after treatment for clinical assessments
and laboratory tests. Previous work demonstrated that participants receiving sham
therapy failed to show changes in immune modulation and metabolic control [18]. Thus, the main outcome measures in current trial were changes in glycated hemoglobin
(HbA1C) values, islet β-cell function of T2D, and immune markers between baseline
and follow-up.

Efficacy measurements in metabolic control

To determine the insulin sensitivity, we used fasting plasma C-peptide instead of
fasting insulin for homeostasis model assessment of insulin resistance (HOMA-IR) and
pancreatic islet β-cell function (HOMA-B) analysis, because 1) C-peptide is a by-product
of insulin synthesis and released at equal levels and 2) T2D patients received external
insulin injections and other treatments that limit the accuracy of HOMA-IR [20,21]. HOMA-IR c-pep was calculated using the equation [20-22]: HOMA-IR c-pep = FPG (mmol/L) × FPC (pmol/L)/22.5. FPG is the value of fasting plasma
glucose. FPC is the value of fasting plasma C-peptide. The denominator of 22.5 is
a normalizing factor [20]. HOMA-B was calculated using the equation [21,22]: HOMA-B c-pep = 20 × FPC (pmol/L)/(FPG (mmol/L)-3.5).

Study end points

The primary study end points were feasibility and safety of the Stem Cell Educator
therapy through 12 weeks post-treatment and preliminary evaluation of the efficacy
of the therapy for change in HbA1C values of T2D through 12 weeks compared to baseline.
Pancreatic islet β cell function was assessed by measuring basal and glucose-stimulated
C-peptide production over time, as described elsewhere [23,24]. Metabolic control was monitored throughout the study. The secondary study end point
was preliminary evidence for efficacy of the therapy in anti-inflammation. Baseline
blood samples were collected prior to Stem Cell Educator therapy.

Cell sorting and co-cultures

To purify CD14+ monocytes, the freshly-isolated peripheral blood mononuclear cells (PBMC) were initially
incubated with 2.5% horse serum to block Fc receptor binding and then incubated with
FITC-conjugated CD14 (eBiosciences) antibody for 45 minutes at 4°C and subjected to
cell sorting using MoFlo (Beckman Coulter, Brea, CA, USA). After confirming the purity
of the population (>98%), CD14+ monocytes were collected and used in different in vitro co-culture experiments with CB-SCs. Culture of CB-SCs were performed as previously
described [18]. Purified CD14+ monocytes were co-cultured with CB-SCs at a ratio of 1:5 of CB-SCs:monocytes. After
co-culture with CB-SCs for 18 hours, floated cells were collected for apoptotic assay
(eBiosciences) by flow cytometry.

To determine the molecular mechanisms underlying the interaction between CB-SCs and
monocytes, blocking experiments with TNF-RI mAb, TNF-RII mAb and inducible nitric
oxide synthase (iNOS) inhibitor 1400W were performed as previously described [15]. Before co-culture with CB-SCs, monocytes were initially stimulated with lipopolysaccharide
(LPS, 10 μg/ml) stimulation for 8 hours, and then seeded onto CB-SCs in regular culture
medium at a ratio of 1:5 of CB-SCs:monocytes for 48 hrs in the presence or absence
of 1400W (100 nM). To block the action of TNF-RI and TNF-RII, the functional grade
purified anti-human TNF-RI and TNF-RII monoclonal antibodies (R&D Systems) were administrated
at 20 μg/ml in 0.1% BSA/PBS buffer. The 0.1% BSA/PBS buffer-treated wells served as
controls. After incubation with CB-SCs at 37°C for two hours, cells were washed with
PBS to remove the unused antibodies. The sorted CD14+ T cells (1 × 105 cells/ml/well) were seeded onto the TNF-RI or TNF-RII antibody-treated wells in duplicate.
To block the action of iNOS and nitric oxide (NO) production, CB-SCs were pre-treated
with 1400W (100 nM, Sigma-Aldrich, St. Louis, MO, USA) for 2 hrs, and then co-cultured
with LPS-stimulated monocytes for 48 hrs, followed by real time PCR analysis by using
Human Th17 for Autoimmunity and Inflammation PCR Array kit (SABiosciences, Valencia,
CA, USA).

Statistical analysis

An intention-to treat approach was used, with 36 patients undergoing Stem Cell Educator
therapy. All patients were included in the safety analyses. The primary efficacy end
point was the change in HbA1C between baseline and follow-up, with an absolute difference
in HbA1C level of at least 0.5% from baseline.

Results

Feasibility and safety of Stem Cell Educator therapy in T2D

Baseline characteristics of participants with T2D are provided in Table 1. Thirty-six patients with T2D have received Stem Cell Educator therapy in a safety
study, and their results are similar to the safety evaluation with T1D participants
[18]. No participants experienced any significant adverse events during the course of
treatment and post-treatment for over a year. Patient complaints were limited to mild
discomfort during venipunctures at the site of median cubital vein and some soreness
of the arm that resolved quickly following aphaeresis.

Efficacy outcomes in improving metabolic control

After receiving Stem Cell Educator therapy and being discharged from the hospital,
patients continued their regular medications. Follow-up studies demonstrated that
the median glycated hemoglobin (HbA1C) in Group A (n = 18) and Group B (n = 11) was significantly lowered from 8.61% ±
1.12 at baseline to 7.9% ± 1.22 at 4 weeks post-treatment (P = 0.026), 7.25% ± 0.58 at 12 weeks post-treatment (P = 2.62E-06) (Figure 1A), and 7.33% ± 1.02 at one-year post-treatment (P = 0.0002). According to the A1C goal (<7%) recommended by the American Diabetes Association
(ADA) for the treatment of adult diabetics, 28% (5/18) of subjects in Group A, 36%
(4/11) of subjects in Group B, and 29% (2/7) of subjects in Group C achieved this
goal at 12 weeks post-treatment. More than 31% of total subjects achieved and maintained
the <7% standard for over a year. Additionally, based on the efficacy criteria, 11
of 18 (61.1%) subjects in Group A, 8 of 11 (72.7%) subjects in Group B, and 4 of 7
(57.1%) subjects in Group C had a reduction of A1C value (>0.5%) at four weeks post-treatment.
Thirteen of 18 (72.2%) subjects in Group A, 9 of 11 (81.8%) subjects in Group B, and
6 of 7 (85.7%) subjects in Group C had a reduction of A1C value (>0.5%). Twenty-eight
of 36 (78%) of the total subjects decreased A1C levels by 1.28 ± 0.66 at 12 weeks
post-treatment. The data demonstrate that glycemic control was improved in T2D patients
after Stem Cell Educator therapy.

To explore the change in insulin sensitivity, we analyzed HOMA-IR by the product of
fasting plasma glucose and C-peptide (instead of insulin due to subjects receiving
insulin injections) in Group A and B. The data revealed that levels of HOMA-IR c-pep
were markedly reduced at four weeks follow-up (Figure 1B). It suggests that insulin sensitivity has been improved post-treatment. Consistent
with their improved β cell function, the median daily dose of metformin was decreased
from 33% to approximately 67%, and insulin was decreased to 35% at 12 weeks post-treatment.

Notably, we found that levels of fasting C-peptide were markedly increased in the
long-standing T2D subjects with impaired islet β cell function (Group C, diabetic
duration 14 ± 6 years, n = 7, P = 0.0073) (Figure 1C). Twelve weeks after receiving the Stem Cell Educator therapy, fasting C-peptide
levels reached normal physiological levels and were maintained through the last follow-up
for this measure (56 weeks) (0.36 ± 0.19 ng/ml at baseline vs 1.12 ± 0.33 ng/ml at
one year post-treatment, P = 0.00045, Figure 1C). The β-cell functional analysis by using HOMA-B C-peptide demonstrates that the
function of islet β cells was markedly enhanced in group C subjects after receiving
Stem Cell Educator therapy (Figure 1D). The data suggest that the restoration of C-peptide may be associated with the
regeneration of islet β cells as we demonstrated in our previous work in type 1 diabetes
[16,18].

Efficacy outcomes in correcting the immune dysfunction

To determine the molecular and cellular mechanisms underlying the improvement of metabolic
control, we examined the effects of anti-inflammation and immune modulation of Stem
Cell Educator therapy in T2D. We used ELISA to examine pro-inflammatory cytokines
IL-1, IL-6 and TNFα in the plasma, which are primarily involved in insulin resistance
and T2D [8,26]. We found that IL-1, IL-6 and TNFα were all at background levels in these long-standing
T2D subjects and failed to show changes after Stem Cell Educator therapy (P = 0.557, P = 0.316, P = 0.603, respectively), probably because metabolic inflammation is a chronic sub-degree
inflammation [8] and the plasma samples which were directly collected from the blood of T2D patients,
not from the lipopolysaccharide (LPS)-activated monocytes of T2D subjects [27]. Importantly, we found that anti-inflammatory and immune suppressive cytokine TGF-β1
was markedly increased in the plasma of T2D subjects post-treatment at four weeks
relative to the baseline levels (Figure 2A). However, IL-10 was unchanged in all participants (P = 0.497). These findings suggest up-regulation of TGF-β1 may be one of potential mechanisms
contributing to the reversal of insulin resistance by Stem Cell Educator therapy.

Figure 2.Anti-inflammatory effects of stem cell educator therapy. (A) Up-regulation of plasma levels of TGF-β1 in T2D patients at baseline and four weeks
after Stem Cell Educator therapy. (B) Flow analysis of intra-cellular cytokines demonstrating differential effects on key
interleukins at four weeks post-treatment. (C) Down-regulation percentage of CD86+CD14+monocytes in T2D patients at baseline and four weeks after Stem Cell Educator therapy.
(D) Flow Analysis of CD4+CD25+Foxp3+ Tregs demonstrating no change in the percentage of Tregs at four weeks post-treatment.

Next, using a more sensitive intra-cellular flow cytometry analysis, we examined interleukin-17
(IL-17, also known as IL-17A) and Th1/Th2 immune response-associated cytokines in
the peripheral blood of T2D subjects. IL-17A is a well-known pro-inflammatory cytokine
involved in autoimmune diseases. Importantly, mounting evidence collected over the
past decade indicates that the etiology of T2D includes an autoimmune component that
initiates an inflammation affecting pancreatic islet β cells [8,28-32], which provides new insight into the mechanism and potential treatment of insulin
resistance through immune modulation. Recent clinical studies showed the increase
of circulating Th17 cells and IL-17 production in T2D patients [33] and obese patients [34]. Additionally, recent studies showed that the level of Th1-associated cytokine IL-12
is increased in T2D subjects [35,36]. We found that the production of IL-17, IL-12 and Th2-associated cytokine IL-4 and
IL-5 were all markedly decreased after Stem Cell Educator therapy (Figure 2B).

To explore the cellular mechanism underlying the modulation on the Th1/Th2 immune
responses, we focused on the changes of co-stimulating molecules CD80/CD86 expressed
on the monocytes/macrophages, the professional antigen-presenting cells that play
a key role in the onset of chronic inflammation and obesity-associated insulin resistance
of T2D [6,37-40]. Flow results demonstrated that the percentage of CD86+CD14+ monocytes was markedly decreased four weeks after treatment (Figure 2C, P = 0.0212). There was no significant change in the level of CD80+CD14+ monocytes (P = 0.13). The ratio of CD86+CD14+ monocytes/CD80+CD14+ monocytes was reduced from 3.86 ± 2.56 to 1.22 ± 0.48 (P = 0.01). Further flow analysis of the ligands of CD80/CD86, CD28/CTLA-4 expressed on
lymphocytes revealed that the expression of CTLA-4 was markedly increased four weeks
after receiving Stem Cell Educator therapy (0.51% ± 0.5 before treatment vs 1.98%
± 0.51 post-treatment, P = 9.02E-05). However, flow analysis failed to show differences in the expression
of co-stimulating molecule CD28 (69.98% ± 14.17 before treatment vs 61.5% ± 10.89
post-treatment, P = 0.225). Additionally, we examined changes in the CD4+CD25+Foxp3+ Tregs population after receiving Stem Cell Educator therapy. Flow analysis did not
identify any differences between baseline and 4 or 12 weeks post-treatment (Figure 2D, P = 0.689). Therefore, these data suggest that Stem Cell Educator therapy may modulate
the Th1/Th2 immune responses through the action of antigen-presenting cells monocytes
rather than Tregs.

In vitro mechanistic studies of the immune modulation of CB-SCs on monocytes

To better understand the immune modulation of CB-SC on monocytes, we performed in vitro co-culture experiments by using CD14+ monocytes purified from human peripheral blood. The purified CD14+ monocytes were co-cultured with CB-SCs at different ratios. We found that there were
strong reactions after adding the CD14+ monocytes to CB-SCs (Figure 3A, bottom left panel). Flow analysis demonstrated that co-culture with CB-SCs for
18 hrs resulted in the significant apoptosis of monocytes at the ratio 1:5 of CB-SC:monocytes
(Figure 3B). Correspondingly, both the cell viability and attachment of CB-SCs were also affected
in the presence of apoptotic monocytes (Figure 3A, bottom left panel). The cellular processes of CB-SCs were reduced in length, but
most were still attached to the bottom (Figure 3A, bottom left panel). Interestingly, these impaired CB-SCs were restored after co-culture
for 2 to 3 days; they continually expanded and became 90 to approximately 100% confluence
after 7 to 10 days (Figure 3A, bottom right panel). Mechanistic studies revealed that CB-SCs displayed the cellular
inhibitor of apoptosis protein (cIAP) 1 [41] that protects CB-SCs against the cytotoxic effects of monocytes, allowing them to
survive and proliferate (Figure 3C). To further explore the molecular mechanisms underlying the cytotoxic effects of
monocytes on CB-SCs, we found that CB-SCs expressed TNF-RII but not TNF-RI (Figure 3D). Recombinant TNF showed cytotoxicity to CB-SCs at different doses (Figure 3E). Notably, CB-SCs pre-treated with TNF-RII mAb (20 μg/ml) at a ratio of 1:10 could
markedly block the toxic action of monocytes and protect 50% of CB-SCs with good cell
viability and morphology.

Figure 3.In vitro study of the immune modulation of CB-SCs on monocytes. (A) Phase contrast microscopy shows the co-culture of CB-SC with monocytes (bottom left
panel) for 18 hrs. CB-SCs co-culture with lymphocytes (top right panel) served as
the control. The impaired CB-SCs after co-culture with monocytes were restored to
expansion and became 90 to approximately 100% confluence after 7 to 10 days (bottom
right). Original magnification, × 100. (B) Apoptotic analysis of floating cells from the co-culture of CB-SCs with monocytes
for 18 hrs. (C) Western blotting shows the expression of the cellular inhibitor of apoptosis protein
(cIAP) 1, not cIAP2, in four preparations of CB-SCs. (D) Western blotting shows the expression of tumor necrosis factor receptor II (TNF-RII),
not TNF-RI, in four preparations of CB-SCs. (E) TNFα suppresses the proliferation of CB-SCs in a dose–response manner. Cell proliferation
was evaluated using CyQUANTR Cell Proliferation Assay Kit [25]. (F) The blocking experiment with iNOS inhibitor 1400W demonstrates that CB-SC-derived
nitric oxide (NO) contributes to the immune modulation of CB-SCs on monocytes. Monocytes
were initially stimulated with lipopolysaccharide (LPS, 10 μg/ml) for 8 hrs, and then
co-cultured with CB-SCs at ratio 1:5 of CB-SCs:monocytes for 48 hrs in the presence
or absence of 1400W (100 nM), followed by real time PCR analysis by using Human Th17
for Autoimmunity and Inflammation PCR Array kit (SABiosciences, Valencia, CA, USA).

To further explore the immune modulation of CB-SCs on monocytes, LPS-stimulated purified
CD14+ monocytes were co-cultured with CB-SCs. Real time PCR array showed that co-culture
with CB-SC could significantly down-regulate numbers of LPS-stimulated, inflammation-related
genes, including chemokines, multiple cytokines and matrix metallopeptidase, along
with signaling pathway molecule NF-κB (Figure 3F). These data clearly indicate that in vitro co-culture with CB-SCs causes substantial down-regulation of inflammation-associated
gene expressions in monocytes. Previous work showed that CB-SCs function as immune
modulators on lymphocytes via nitric oxide (NO) production [15]. To confirm the action of NO involved in the immune modulation of CB-SCs on monocytes,
the specific inducible nitric oxide synthase (iNOS) inhibitor 1400W was applied to
the co-culture system. The data demonstrated that the inhibitory effects of CB-SC
on LPS-stimulated monocytes could be significantly reversed in the presence of iNOS
inhibitor 1400W (Figure 3F). Interestingly, we found that blocking NO production in CB-SCs could markedly increase
the expressions of chemokine CCL20 and cytokines (for example, IL-1β, IL-6, IL-8,
IL-23 and TNFα) in monocytes. Thus, it indicates that CB-SC-derived NO plays an essential
role in the immune modulating and anti-inflammatory effects of CB-SCs on monocytes.

Discussion

Insulin resistance is the hallmark of T2D. It is widely accepted that the inability
of pancreatic β cells to function in compensating for insulin resistance leads to
the onset of clinical diabetes. Persistent metabolic stresses including glucotoxicity,
lipotoxicity, chronic metabolic inflammation, oxidative stress and endoplasmic reticulum
stress, cause progressive dysfunction of islet β cells and finally lead to the cellular
death and absolute shortage of islet β cells in long-standing T2D subjects [42]. The current phase 1/2 study demonstrates the safety and therapeutic efficacy of
Stem Cell Educator therapy in the treatment of T2D. Insulin sensitivities were markedly
increased after receiving Stem Cell Educator therapy, followed by the significant
improvement of metabolic controls in these long-standing T2D patients. Notably, we
found that T2D subjects in Group C (with the absolute shortage of islet β cells) significantly
improved fasting C-peptide levels and β cell function. These data indicate that Stem
Cell Educator therapy may open up a new avenue for the treatment of T2D.

Chronic inflammation of visceral adipose tissue (VAT) is a major contributor to insulin
resistance mediated by adipose tissue-released adipokines (for example, IL-6, TNFα,
MCP-1 and resistin) [40,43]. Growing evidence strongly demonstrated that an accumulation of macrophages by metabolic
stress in the sites of affected tissues (such as vasculature, adipose tissue, muscle
and liver) has emerged as a key process in the chronic metabolic-stress-induced inflammation
[44]. Monocytes/macrophages, as one type of the professional antigen-presenting cells,
play an essential role in controlling the Th1/Th2 immune responses and maintaining
homeostasis through the co-stimulating molecules CD80/CD86 and released cytokines.
Persistent destructive effects of lipid influx (for example, fatty acids and cholesterol)
cause macrophage dysfunctions (including defective efferocytosis and unresolved inflammation),
resulting in recruitment and activation of more monocytes/macrophages via MCP-1 and
its receptor CCR2 [44]. Consequently, inflammatory cytokines (for example, IL-6 and TNFα) produced by activated
macrophages induce insulin resistance in major metabolic tissues [26,44,45]. To prove the action of macrophage in chronic inflammation and insulin resistance
in T2D, conditional depletion of CD11c+ macrophages or inhibition of macrophage recruitment via MCP-1 knockout in obese mice
resulted in a significant reduction in systemic inflammation and an increase in insulin
sensitivity [46-48].

To clarify the modulation of Stem Cell Educator therapy on blood monocytes, we found
that expression of CD86 and CD86+CD14+/CD80+CD14+ monocyte ratios have been markedly changed after receiving Stem Cell Educator therapy
in T2D subjects. CD80 and CD86 are two principal co-stimulating molecules expressed
on monocytes to skew the immune response toward Th1 or Th2 differentiation through
their ligands CD28/CTLA4 [49,50]. Due to the differences of expression levels and binding affinity between CD80 and
CD86 with their ligands CD28/CTLA4, it is widely accepted that the interaction of
CD86 with CD28 dominates in co-stimulating signals; conversely, the combination of
CD80 and CTLA4 governs negative signaling [49-52]. The normalization of the CD86+CD14+/CD80+CD14+ monocyte ratio post-treatment may favor the immune balance of Th1/Th2 responses in
diabetic subjects. Taken together with our in vitro study on the direct interaction between CB-SCs and purified CD14+ monocytes, these data indicate that restoration of monocyte functions (such as the
expression of CD86, cytokine productions and chemokine productions) mainly contributes
to anti-inflammation and reversal of insulin resistance following Stem Cell Educator
therapy in T2D subjects.

Increasing animal and clinical evidence demonstrate multiple immune cells contributing
to the inflammation-induced insulin resistance in T2D, such as abnormalities of lymphocytes
(including T cells, B cells and Tregs [53-57]), neutrophils [58], eosinophils [59], mast cells [60] and dendritic cells (DCs) [61,62]. Specifically, B and T lymphocytes have emerged as unexpected promoters and controllers
of insulin resistance [57]. These adaptive immune cells infiltrate into the VAT, releasing cytokines (IL-6 and
TNFα) and recruiting more monocytes/macrophages via MCP-1/CCR2 [44]. Finally, this obesity-related inflammation leads to insulin resistance [57,63]. Thus, a major challenge for treatment of T2D is to identify therapeutic approaches
that fundamentally correct insulin resistance through targeting the dysfunctions of
multiple immune cells. The valuable lessons from intensive research pressure over
the past 25 years in T1D [11] highlight the difficulties in overcoming these multiple immune dysfunctions by utilizing
conventional immune therapy. Stem Cell Educator therapy functions as “an artificial
thymus” that circulates a patient’s blood through a blood cell separator [19], briefly co-cultures the patient’s blood mononuclear cells (such as T cells, B cells,
Tregs, monocytes and neutrophils) with CB-SCs in vitro. During the ex vivo co-culture in the device, these mononuclear cells can be educated by the favorable
microenvironment created by CB-SCs through: 1) the action of an autoimmune regulator
(AIRE) expressed in CB-SCs [18]; 2) the cell-cell contacting mechanism via the surface molecule programmed death
ligand 1 (PD-L1) on CB-SCs [15]; and 3) the soluble factors released by CB-SCs. Previous work [15] and current data indicate that CB-SC-derived NO mainly contributes to the immune
modulation on T cells and monocytes. During the passage of monocytes and other immune
cells through the device, NO, as a free radical released by CB-SCs, can quickly transmit
into their cellular membrane, without the aid of dedicated transporters; 4) correcting
the functional defects of regulatory T cells (Tregs) [16]; and 5) directly suppressing the pathogenic T cell clones [17]. During this procedure, both peripheral and infiltrated immune cells in VAT can be
isolated by a blood cell separator and treated by CB-SCs, leading to the correction
of chronic inflammation, the restoration of the immune balance, and clinical improvements
in metabolic control via increasing of insulin sensitivity. Additionally, TGF-β1 is
a well-recognized cytokine with a pleiotropic role in immune modulation on multiple
immune cells, such as the differentiation and function of Th1/Th2 cells and Tregs,
as well as B cells, monocytes/macrophages, dendritic cells, granulocytes and mast
cells [64-66]. These immune cells are involved in the inflammation-induced insulin resistance in
T2D [53-62]. Therefore, the up-regulation of TGF-β1 level in peripheral blood of T2D subjects
is another major mechanism underlying the immune modulation after receiving Stem Cell
educator therapy.

During the procedure of Stem Cell Educator therapy, the mononuclear cells circulating
in a patient’s blood are collected by a blood cell separator. Additionally, patients
are required to move their hips, legs and turn to one side every 15 to 30 minutes
during the treatment, in order to mobilize their immune cells from peripheral tissues
(including adipose tissues) and organs entering into the blood circulation to be processed
by a blood cell separator. Thus, the immune cells both in peripheral blood and in
tissues can be isolated by a blood cell separator and treated by CB-SCs. The full
blood volume is processed approximately twice during Stem Cell Educator therapy (approximately
10,000 ml whole blood) [18], which ensures a comprehensive approach to modulating essentially all circulating
immune cells to address multiple immune dysfunctions and overcome global insulin resistance
resulting from a variety of reasons. No other current medications and/or other approaches
have yet been shown to achieve this unique therapy success. There are some pathogenic
immune cells remaining in tissues and lymph nodes which fail to enter into the blood
circulation during the procedure and may escape from the treatment by CB-SCs. These
immune cells may migrate into the blood circulation and decrease the therapeutic effectiveness.
Therefore, T2D subjects may need additional treatment six to nine months later after
receiving the first treatment; however, this is yet to be explored in the phase 3
clinical trial.

We observed that the improvement of islet β cell function (C-peptide levels) progresses
slowly over weeks after receiving Stem Cell Educator therapy, not disappearing with
the progression of time. We reported similar data in previous T1D trials [18,19]. If Stem Cell Educator therapy only temporarily corrects the immune dysfunctions,
the clinical efficacy in metabolic control should disappear soon after receiving Stem
Cell Educator therapy, because of the short lifespans of most immune cells, (for example,
5.4 days for neutrophils [67], 3 months for lymphocytes, 1 to 3 days for bone marrow-derived monocytes existing
in blood and then migrating into tissues). Previous work demonstrated that CB-SCs
showed the marked modulation of Th1-Th2-Th3 cell-related genes, including multiple
cytokines and their receptors, chemokines and their receptors, cell surface molecules,
along with signaling pathway molecules and transcription factors, as indicated by
quantitative real time PCR array [16]. Due to these fundamental immune modulations and induction of immune balance [19], this trial indicates that a single treatment with Stem Cell Educator therapy can
give rise to long-lasting reversal of immune dysfunctions and improvement of insulin
sensitivity in long-standing T2D subjects.

Conclusions

The epidemic of diabetes is creating an enormous impact on the global economy, as
well as on the health of humans. Overcoming insulin resistance is a major target for
the treatment of T2D, and mounting evidence points to the involvement of multiple
immune dysfunctions in T2D [3,37,40]. Monocytes/macrophages act as key players contributing to these chronic inflammations
and leading to insulin resistance in T2D [6,33,37,39,40]. The current phase 1/phase 2 study demonstrates that Stem Cell Educator therapy can
control the immune dysfunctions and restore the immune balance through the modulation
of monocytes/macrophages and other immune cells, both in peripheral blood and in tissues,
leading to a long-lasting reversal of insulin resistance and a significant improvement
in insulin sensitivity and metabolic control in long-standing T2D subjects. These
findings are subject to further investigation in large-scale, multi-center clinical
trials. This novel approach holds great promise for improving treatment and finding
a cure for diabetes, specifically for early-stage diabetics. The advantages of Stem
Cell Educator therapy may help diabetics to cope with diabetes-associated complications
and improve their quality of life.

Competing interests

Dr. Zhao, inventor of this technology, led the clinical study and has an investment
and a fiduciary role in Tianhe Stem Cell Biotechnology, Inc. (and licensed this technology
from University of Illinois). YeZ, SW, JS and YL are employees of Tianhe Stem Cell
Biotechnologies, Inc., which might have an interest in the submitted work. All other
authors (ZJ, TZ, MY, CH, HZ, ZY, YC, HT, SJ, YD, YiC, XS, MF and HL) have no financial
interests that may be relevant to the submitted work.

Authors’ contributions

YZ and ZJ designed the trial and analyzed the data. YZ drafted the manuscript and
obtained the funding. ZY, YeZ, HT and SJ collected data. TZ, MY, CH, HZ, YaC, SW,
JS, YL, YD, YC, XS, MF and HL contributed to administrative, technical or material
support. All authors had full access to all the data and take responsibility for the
integrity of the data and the accuracy of the data analysis. All authors read and
approved the final manuscript.

Acknowledgements

This clinical trial was supported by the China Jinan 5150 Program, Jinan High-Tech
Development Zone, Ministry of Human Resources and Social Security of the P.R. China.
Ex vivo mechanistic studies were supported by the American Diabetes Association. Sponsors
had no role in conception, design or conduct of the study; collection, management,
analysis or interpretation of the data; or the preparation, review or approval of
the manuscript. The researchers worked independently of the funders.